The present invention relates to assays for the determination of analytes in fluids and, more particularly, to a method for correcting the signal obtained for variations caused by relative humidity and/or temperature variations.
Many types of assay elements for the rapid analysis of analytes present in biological fluids are known in the art. Of particular interest are dry multilayer analytical elements to which a sample, e.g., a drop of blood, serum or plasma, is applied and allowed to migrate or diffuse to a reagent layer or layers. As a result of the interaction between the analyte and the reagent(s) present, a detectable change is brought about in the element corresponding to the presence of the analyte in the sample. The detectable change can be a color change which may be evaluated visually or read spectrophotometrically such as with a densitometer. In another scheme based on the presence of fluorescent-labeled biologically active species, a fluorescent output signal can be generated and read spectrofluorometrically. Such assay elements are of great interest because they can be adapted for use in automated analytical instruments.
In the automated analytical instruments a sample of a test fluid is typically provided in a sample cup and all of the assay method steps including pipetting of a measured volume of the sample onto an assay element, incubation and readout of the signal obtained as a result of the interaction(s) between the reagent(s) and the sample analyte are carried out automatically. The assay element is typically transported from one station, e.g., the pipetting station, to another, e.g., the optical read station, by a transport means such as a rotating carousel to enable the test steps to be carried out automatically. Further, in some instruments the pipetting and optical read steps are carried out while the assay element is in a temperature-controlled chamber. Such automated instruments can be operated in the batch mode, that is, a plurality of assays for the same analyte are carried out at the same time. Also, some instruments are capable of being operated in a random-access mode, that is, assays for a plurality of different analytes can be carried out at the same time.
Such automated analytical instruments are capable of processing many assay elements rapidly and it is necessary to achieve a very high level of precision for these assays. However, imprecisions in the results obtained can be caused by a number of factors. Commonly assigned, copending U.S. patent application Ser. No. 382,555, filed Jul. 19, 1989 now U.S. Pat. No. 5,156,079 discloses and claims an analytical method which utilizes dry multilayer assay elements. The method corrects for signal imprecisions caused by variations in reagent levels from assay element to assay element and other variations including those in instrument position response. Briefly, this method involves taking a first optical reading of the assay element prior to delivering sample fluid to the element and, after the fluid has been dispensed and the requisite interaction between the sample analyte and the reagent(s) has taken place, taking a second optical reading. The ratio of the second signal to the first signal is taken and compared with that for known amounts of the analyte to determine the amount of analyte in the fluid.
As the state of the art advances, however, additional difficulties may be encountered. For example, the optical signals obtained from various dry assay elements including multilayer assay elements can vary significantly depending upon the relative humidity of the environment in which the instrument is located. Typically, these diagnostic assay elements are packaged in moisture impermeable materials and the relative humidity inside the package maintained at a very low level, e.g., about 10%, so as to prolong the shelf life of the elements. The assay elements should be removed from the package just prior to use and inserted into the analytical instrument without being allowed to remain in the ambient environment for any extended length of time. However, it is apparent that the ambient environment will vary from location to location and also within a location over time. Therefore the relative humidity within the instrument will vary.
Dealing with relative humidity and temperature considerations in random access analyzers involves still another level of difficulty since the individual assay elements may reside in the instrument for different periods of time before the dry optical reading is taken. One approach to dealing with temperature considerations in such analyzers is to allow the assay element to remain in a temperature controlled chamber for the period of time necessary for the element to equilibrate with the chamber temperature. A possible approach to the relative humidity factor would be to take the dry reading only after a particular assay element has been in the instrument for a period of time sufficient to permit it to come to equilibrium with the relative humidity of the environment and to correct the reading based on the relative humidity as measured by a sensor. Such procedures are not entirely satisfactory because imposing such minimum time periods on the assay protocols would restrict the throughput rate of the instrument and would not permit the instrument to be operated in the most efficient manner.
It would be desirable therefore to have a method for compensating for optical signal imprecisions caused by relative humidity and/or temperature variations which offers a number of optional choices based on the manner in which the operator desires to practice the method. It would also be desirable to have such a method which can be practiced so as not to adversely affect the throughput rate of the analytical instrument. Further, it would be desirable to have such a method which would permit a random access automated analytical instrument to be operated in the most efficient manner possible.